PROJECT 3: Quality of T-Cell Responses Following DENV Natural Infections and Live-Attenuated Dengue Virus Vaccination (La Jolla Institute for Allergy and Immunology) SUMMARY The overall theme of the P01 is to study and reveal the role of adaptive immunity in the context of DENV infection and vaccination, in terms of shaping clinical and virological endpoints and outcomes. In Project 3, we will specifically address the central issue of the relation between quality of T cell responses and clinical outcomes as well as antibody quality and durability. Beyond accurately measuring magnitude and number of DENV-specific T cells, we will thoroughly define the quality of T cell responses. First, we will study the antigens targeted by DENV-specific T cells; for example, are envelope (E)-specific T cell responses more effective for helping B cells? Are non-structural (NS) protein-derived responses better for providing help for killing infected targets by CD8 T cells? Second, we will examine another important component of response quality, namely its cross-reactivity among DENV serotypes, or with the vectors used in vaccination. This issue is of relevance in the context of the suboptimal performance of the first licensed DENV vaccine (Dengvaxia), which utilizes DENV-derived E proteins expressed in a yellow fever virus backbone, which lacks DENV NS proteins. Third, we will determine the CD4 and CD8 subsets elicited by infection and vaccination, in terms of which memory subsets drive responses, what their specific transcriptomic profiles are, to what degree effector responses are multi-specific, and how narrow or diverse the T cell receptor (TCR) repertoire of DENV-specific T cell is. This comprehensive analysis will allow to ask the questions: What drives quality? And how does quality influence outcomes? Our hypothesis is that the quality of T cell responses influences the clinical outcomes of infection and vaccination, either by direct effector functions of DENV-specific T cells, or indirectly, through modulation of the quantity and quality of B cell responses. This hypothesis will be tested by characterizing the quantity and quality of memory T cell responses prior to either symptomatic (DF or severe dengue disease) or inapparent DENV infection in samples distributed by Core C from the Pediatric Dengue Cohort Study in Nicaragua (Aim 1). We will also investigate the role of T cell help in relation to antibody quality and durability (Project 1). Further, we will utilize samples collected 12-18 month after Dengvaxia vaccination in the Philippine cohort (Core C), and specifically focus on differences observed in DENV-seronegative versus -seropositive vaccinees prior to vaccination (Aim 2). In parallel, we will analyze samples from vaccinees who experience breakthrough DENV infections after vaccination. The variables considered to define ?quality? will include: magnitude of antigen- specific T cell response, definition of the dominant antigenic targets, cross-reactivity with other serotypes, determination of memory and CD4/CD8 subsets, multi-specificity in terms of cytokine secretion, transcriptomic profiles, and TCR repertoire diversity. We will test which of these ?quality? measures, either alone or in combination with other variables, best correlates with the clinical outcomes. These studies are closely linked with other projects in this P01, as Project 1 (natural infection) and Project 2 (vaccination) will use the same samples/sample sets to investigate the quality of humoral responses. Statistical modeling as well as integrated analyses across projects will be performed by Core B. Overall, our studies will establish correlates of protection from DENV disease based on quality of T cell responses, which will constitute important contributions to dengue vaccine development and evaluation.